The long-term goal of our research is to understand the molecular mechanisms by which neurons acquire their characteristic pattern of connectivity during development. Neurons are morphologically diverse cells that are specialized for communication with their elaborate dendrites designed to receive, and axons to send, information. These processes and the specificity by which they connect to each other define the functional nervous system. While recent work has demonstrated that directed growth of axons is determined by the extracellular cues, much is to be learned about how the growth cone interprets and translates these signals into fine reorganization of the cytoskeleton. Small GTP-binding proteins of the Rho subfamily, including Rho, Rac, and Cdc42, regulate the actin cytoskeleton from yeast to humans. We have previously shown that they play important and distinct roles in the morphogenesis of neurons in both flies and mammals. Our working hypothesis is that these GTP-binding proteins receive signals from cell surface receptors and in turn regulate reorganization of the cytoskeleton necessary for growth cone motility and guidance. We have recently established a genetic mosaic method to study the function of these ubiquitously expressed and pleiotropically functional genes in small populations of identified neurons in the Drosophila brain. In this application we propose to systematically investigate how Rho, Rac and Cdc42 regulate the neuronal cytoskeleton by studying genetic loss- and gain-of-function mutants in these Rho GTPases and their effector pathway components. We will also explore the potential "cross talk" between these downstream effector pathways. To investigate the functions of these genes, we will use several in vivo paradigms that we have characterized, in combination with in vitro cultures. Our studies will shed light on the role of each of the Rho GTPases, how they transduce signals to the cytoskeleton, and how these signal transduction pathways interact with each other to bring about the myriad of morphological changes regulated by the Rho GTPases. Mutations in the Rho GTPase signaling pathway components have been reported in many human diseases. These include human neurological diseases such as non-syndromic mental retardation and William's syndrome, underscoring the importance of these signaling molecules in the development and function of the nervous system, and in human mental health.